The present invention relates to a modified tRNA molecule which inhibits the full length reverse transcription of HIV-1 and HIV-1 replication. The invention further relates to a method for inhibiting the replication of HIV-1 in human cells.
A retrovirus designated human immuno-deficiency virus (xe2x80x9cHIVxe2x80x9d) is the causative agent of the complex disease termed Acquired Immune Deficiency Syndrome (xe2x80x9cAIDSxe2x80x9d) and is a member of the lentivirus family of retroviruses. M. A. Gonda, et al., Science 227, 173, (1985); P. Sonigo, et al., Cell 42, 369, (1985). The complex disease AIDS includes progressive destruction of the immune system and degeneration of the central and peripheral nervous systems. The HIV virus was previously known or referred to as LAV, HTLV-III, or ARV. The Centers for Disease Control (xe2x80x9cCDCxe2x80x9d) reported the number of AIDS deaths for the first six months of 1996 was 22,000 nationwide.
tRNA functions in living cells mainly as a vehicle to translate genetic information stored in mRNA into amino acid sequence in proteins. Cellular tRNAs are recognized by many cellular proteins including 5xe2x80x2 and 3xe2x80x2 tRNA processing enzymes (Altman, S., Cell 23, 1-5 (1981)) and tRNA aminoacyl transferases (aa-tRNA synthetase) (Sampson, J. R., et al., Biochemistry 29, 2523-2532 (1990); Behlen, L. S., et al., Biochemistry 29, 2515-2523 (1990)). Most of these enzymes recognize both the anticodon region and specific features of the tridimensional structure of tRNA (Sampson, J. R., et al., Biochemistry 29, 2523-2532 (1990)). There are two different human tRNALys genes. One of these, tRNAlys-UUU (Roy, K. L., et al., Nuc. Acids Res. 10, 7313-7322 (1982); Richter-Cook, N. J., et al., J. Biol. Chem. 267, 15952-15957 (1992)), has a complementary sequence to the HIV-1 RNA genome in the region of the prime binding sequence (PBS), and is used as the primer for HIV-1 reverse transcription.
Human immunodeficiency virus type-1 (xe2x80x9cHIV-1xe2x80x9d) relies on multiple human cellular factors for its own replication (Steffy, K., et al., Microbiol Rev 55(2), 193-205 (1991)). In particular, tRNALys3 is utilized by HIV-1 at the earliest step of its life cycle (Weiss, S., et al., Gene 111(2), 183-97 (1992), Das, A. T., et al., FEBS Lett. 341, 49-53 (1994); Arts, E., et al., J. Biol. Chem. 269, 14672-14680 (1994); Barat, C., et al., Embo J 8(11), 3279-85 (1989); Barat, C., et al., Nucleic Acids Res 19(4), 751-7 (1991); Khan, R., et al., J Biol Chem 267(10), 6689-95 (1992)). During the first step of reverse transcription, the single stranded plus-sense RNA genome is copied into minus-sense cDNA beginning at the 3xe2x80x2 end of a partially annealed tRNALys3 primer (Peliska, J. A., et al., Science 258, 1112-1118 (1992)). Several reports indicated that p66 of the HIV-1 RT p51/p66 heterodimer recognizes and binds to the tRNALys3 anticodon region (DeVico, A. L., et al., J. Biol. Chem. 266, 6774-6779 (1991); Sarih-Cottin, L., et al., J Mol Biol 226(1), 1-6 (1992); Rhim, H., et al., J Virol 65(9), 4555-64 (1991); Kohlstaedt, L. A., et al., Proc. Natl. Acad. Sci. USA 89, 9652-9656 (1992)) and may help unwind the acceptor stem (Kohlstaedt, L. A., et al., Proc. Natl. Acad. Sci. USA 89, 9652-9656 (1992)) in the presence of NCp7 protein (Barat, C., et al., Nucleic Acids Res 19(4), 751-7 (1991)). Another report demonstrated that excess wild type tRNALys3 primer inhibited the DNA polymerase activity of a recombinant HIV-1 RT, p66/p51 heterodimeric form (Bordier, B., et al., Nucleic Acids Res 18(3), 429-36 (1990)). This effect was ascribed to the anticodon region of tRNALys3 primer (Bordier, B., et al., Nucleic Acids Res 18(3), 429-36 (1990)). The necessary role for tRNALys3 in HIV-1 reverse transcription, its specific affinity for HIV-1 RT, and its association with HIV-1 virions suggests that mutated derivatives of the tRNALys3 primer might interfere with the viral replication cycle.
HIV-1 specific ribozymes, antisense RNA, and RNA decoys have been proposed as potential therapeutic reagents for HIV-1 (Chatterjee, S., et al., Science 258, 1485-1488 (1992); Sullenger, B. A., et al., Cell 63, 601-608 (1990); Ojwang, J. O., et al., PNAS 89, 10802-10806 (1992)).
Presently, a triple-drug therapy regimen is the most effective approach to controlling the AIDS virus. The cocktail therapy consists of a combination of a protease inhibitor called indinavir with two reverse-transcriptase inhibitors known as AZT and 3TC. The triple drug therapy results in a decrease in measured levels of virus in both blood and lymphatic tissues. However, a proportion of those treated, whose viruses had developed resistance to one or more of the cocktail""s reverse-transcriptase inhibitors as a result of previous treatment, fail to respond. Some studies have projected that combination therapy can virtually eliminate the AIDS virus from those patients who respond within two or three years. However, other studies indicate that minimal residual virus is sufficient to cause relapse. In addition, existing pharmacologic therapies leave residual HIV genes lurking in cells in the form of latent xe2x80x9cprovirus.xe2x80x9d As long as the cells remain alive, these genes can be transcribed, rekindling virus production. Therefore, drug therapy may need to be maintained throughout the patient""s life. The therapy may have long term adverse side effects and treatments can cost over $15,000 annually for each patient. Thus, there is a continuing need to develop additional approaches to controlling HIV infection which can be used alone or in combination with existing therapies.
One aspect of the present invention relates to a modified tRNALys3 molecule for inhibiting HIV template replication. The tRNA molecule has an acceptor stem having 3xe2x80x2 and 5xe2x80x2 segments which form a secondary structure. Furthermore, the 3xe2x80x2 segment hag a nucleic acid sequence with reduced complementarity to the HIV-1 primer binding site compared to the 3xe2x80x2 segment of a wild type tRNALys3. The 5xe2x80x2 segment has sufficient homology with the 3xe2x80x2 segment to maintain the secondary structure of the acceptor stem.
Another aspect of the present invention relates to a tRNALys3 molecule which is modified to inhibit the interaction of the tRNALys3 molecule with HIV-1 reverse transcriptase or HIV-1 RNA template. In this aspect of the invention, the tRNALys3 molecule is modified in a region other than its acceptor stem.
The present invention also provides a method of inhibiting HIV infectivity in human cells by introducing the modified tRNALys3 molecules of the invention into human cells.